Summary An important task of genome duplication is to overcome the large number of diverse obstacles posed by template lesions or DNA and protein blocks. Studies in the past decade suggest the existence of multiple mechanisms to rescue and complete replication in the face of these impediments; these include stabilization and remodeling of stalled replisomes to allow replication resumption, coordination of various types of DNA repair, and utilization of specialized DNA polymerases. Defects in these processes lead to debilitating human pathologies such as sterility, cancer, and developmental defects; thus studying these processes and understanding the underlying mechanisms, which are the goals of our studies, are of considerable significance to human health. Recent studies have shown that the evolutionarily conserved Smc5/6 complex is essential for growth and critical for promoting replication under normal and DNA damaging conditions. We have made significant progress in the first cycle of this grant in revealing the function and structure of this complex in the budding yeast model system. Results from our lab and others suggest that the Smc5/6 complex plays multiple roles in promoting replication; these include the prevention of accumulation of toxic recombination intermediates, the modification of replication and repair proteins with the small protein modifier SUMO, and collaboration with other replication regulatory factors. Currently how the Smc5/6 complex carries out these functions is not well understood, and research into these will shed light on the various replication-promoting processes. In the next grant period, we propose to elucidate the basis of the Smc5/6 complex function in replication using a combination of genetic, biochemical, and genomic approaches in the yeast model system. In Aim 1, we will investigate how the Smc5/6 complex collaborates with another conserved replication regulatory factor Rtt107 to promote replication. We will use quantitative genome-wide measurements of DNA synthesis and replisome-DNA association to characterize the effects of these factors on replication. We will also examine the physical interactions between the Smc5/6 complex, Rtt107, and replication machinery. In Aim 2, we will investigate how the Smc5/6 complex utilizes its SUMO ligase enzymatic activity to influence replication and repair. We have identified several key replication and repair enzymes whose sumoylation shows dependence on this complex. We will examine how the sumoylation of these substrates contributes to replication under different types of stress conditions. In Aim 3, we will investigate the mechanisms that target the Smc5/6 complex and Rtt107 to stalled replication forks to enable replication regulation. These proposed studies have broad implications for understanding how replication is regulated to enable proper development and survival of all organisms.